Tumor associated peptide and uses thereof

ABSTRACT

The invention relates to a peptide with the sequence of RLLEFYLAM, methods for the use of the peptide, and antisera and monoclonal antibodies against the peptide. The peptide is derived from the NY-ESO-1 molecule, which form complexes with HLA molecules, leading to lysis of cells presenting these complexes, by cytolytic T lymphocytes.

RELATED APPLICATION

This application is a divisional application of application Ser. No.09/409,455 filed Sep. 30, 1999 now abandoned, which is acontinuation-in-part of application Ser. No. 09/344,040 filed Jun. 25,2999 now U.S. Pat. No. 6,548,064 which is a continuation-in-part ofapplication Ser. No. 09/105,839 filed Jun. 26, 1998, now U.S. Pat. No.6,287,756.

FIELD OF THE INVENTION

This invention relates to the isolation and cloning of genes which aremembers of the “SSX” family, which is discussed herein, and the usesthereof, including determination of cancer. Also a part of the inventionare peptides derived from these SSX genes, as well as from the NY-ESO-1gene. These peptides stimulate proliferation of cytolytic T cells, andthus are useful as markers for presence of disorders such as cancer, forHLA-A2 cells, and as therapeutic agents for treating cancer.

BACKGROUND AND PRIOR ART

It is fairly well established that many pathological conditions, such asinfections, cancer, autoimmune disorders, etc., are characterized by theinappropriate expression of certain molecules. These molecules thusserve as “markers” for a particular pathological or abnormal condition.Apart from their use as diagnostic “targets,” i.e., materials to beidentified to diagnose these abnormal conditions, the molecules serve asreagents which can be used to generate diagnostic and/or therapeuticagents. A by no means limiting example of this is the use of cancermarkers to produce antibodies specific to a particular marker. Yetanother non-limiting example is the use of a peptide which complexeswith an MHC molecule, to generate cytolytic T cells against abnormalcells.

Preparation of such materials, of course, presupposes a source of thereagents used to generate these. Purification from cells is onelaborious, far from sure method of doing so. Another preferred method isthe isolation of nucleic acid molecules which encode a particularmarker, followed by the use of the isolated encoding molecule to expressthe desired molecule.

To date, two strategies have been employed for the detection of suchantigens, in e.g., human tumors. These will be referred to as thegenetic approach and the biochemical approach. The genetic approach isexemplified by, e.g., dePlaen et al., Proc. Natl. Sci. USA 85: 2275(1988), incorporated by reference. In this approach, several hundredpools of plasmids of a cDNA library obtained from a tumor aretransfected into recipient cells, such as COS cells, or intoantigen-negative variants of tumor cell lines. Transfectants arescreened for the expression of tumor antigens via their ability toprovoke reactions by anti-tumor cytolytic T cell clones. The biochemicalapproach, exemplified by, e.g., Mandelboim, et al., Nature 369: 69(1994) incorporated by reference, is based on acidic elution of peptideswhich have bound to MHC-class I molecules of tumor cells, followed byreversed-phase high performance liquid chromography (HPLC). Antigenicpeptides are identified after they bind to empty MHC-class I moleculesof mutant cell lines, defective in antigen processing, and inducespecific reactions with cytotoxic T-lymphocytes. These reactions includeinduction of CTL proliferation, TNF release, and lysis of target cells,measurable in an MTT assay, or a ⁵¹Cr release assay.

These two approaches to the molecular definition of antigens have thefollowing disadvantages: first, they are enormously cumbersome,time-consuming and expensive; second, they depend on the establishmentof cytotoxic T cell lines (CTLs) with predefined specificity; and third,their relevance in vivo for the course of the pathology of disease inquestion has not been proven, as the respective CTLs can be obtained notonly from patients with the respective disease, but also from healthyindividuals, depending on their T cell repertoire.

The problems inherent to the two known approaches for the identificationand molecular definition of antigens are best demonstrated by the factthat both methods have, so far, succeeded in defining only very few newantigens in human tumors. See, e.g., van der Bruggen et al., Science254: 1643-1647 (1991); Brichard et al., J. Exp. Med. 178: 489-495(1993); Coulie, et al., J. Exp. Med. 180: 35-42 (1994); Kawakami, etal., Proc. Natl. Acad. Sci. USA 91: 3515-3519 (1994).

Further, the methodologies described rely on the availability ofestablished, permanent cell lines of the cancer type underconsideration. It is very difficult to establish cell lines from certaincancer types, as is shown by, e.g., Oettgen, et al., Immunol. Allerg.Clin. North. Am. 10: 607-637 (1990). It is also known that someepithelial cell type cancers are poorly susceptible to CTLs in vitro,precluding routine analysis. These problems have stimulated the art todevelop additional methodologies for identifying cancer associatedantigens.

One key methodology is described by Sahin, et al., Proc. Natl. Acad.Sci. USA 92: 11810-11913 (1995), incorporated by reference. Also, seeU.S. patent application Ser. No. 08/580,980, and filed on Jan. 3, 1996,and U.S. Pat. No. 5,698,396. All three of these references areincorporated by reference. To summarize, the method involves theexpression of cDNA libraries in a prokaryotic host. (The libraries aresecured from a tumor sample). The expressed libraries are thenimmunoscreened with absorbed and diluted sera, in order to detect thoseantigens which elicit high titer humoral responses. This methodology isknown as the SEREX method (“Serological identification of antigens byRecombinant Expression Cloning”). The methodology has been employed toconfirm expression of previously identified tumor associated antigens,as well as to detect new ones. See the above referenced patentapplications and Sahin, et al., supra, as well as Crew, et al., EMBO J144: 2333-2340 (1995).

The SEREX methodology has been applied to esophageal cancer samples, andan esophageal cancer associated antigen has now been identified, and itsencoding nucleic acid molecule isolated and cloned, as per U.S. patentapplication Ser. No. 08/725,182, filed Oct. 3, 1996, incorporated byreference herein.

The relationship between some of the tumor associated genes and a triadof genes, known as the SSX genes, is under investigation. See Sahin, etal., supra; Tureci, et al., Cancer Res 56:4766-4772 (1996). One of theseSSX genes, referred to as SSX2, was identified, at first, as one of twogenes involved in a chromosomal translocation event (t(X; 18)(p11.2; q11.2)), which is present in 70% of synovial sarcomas. See Clark, et al.,Nature Genetics 7:502-508 (1994); Crew et al., EMBO J 14:2333-2340(1995). It was later found to be expressed in a number of tumor cells,and is now considered to be a tumor associated antigen referred to asHOM-MEL-40 by Tureci, et al, supra. Its expression to date has beenobserved in cancer cells, and normal testes only. Thus parallels othermembers of the “CT” family of tumor antigens, since they are expressedonly in cancer and testis cells. Crew et al. also isolated and clonedthe SSX1 gene, which has 89% nucleotide sequence homology with SSX2.Sequence information for SSX1 and SSX2 is presented as SEQ ID NOS: 1 and2 respectively. See Crew et al., supra. Additional work directed to theidentification of SSX genes has resulted in the identification of SSX3,as is described by DeLeeuw, et al., Cytogenet. Genet 73:179-183 (1996).The fact that SSX presentation parallels other, CT antigens suggested tothe inventors that other SSX genes might be isolated. The parentapplication, supra discloses this work, as does Gure, et al. Int. J.Cancer 72:965-971 (1997), incorporated by reference.

With respect to additional literature on the SSX family, most of itrelates to SSX1. See PCT Application W/96 02641A2 to Cooper, et al,detailing work on the determination of synovial sarcoma viadetermination of SSX1 or SSX2. Also note DeLeeuw, et al. Hum. Mol. Genet4(6):1097-1099 (1995). also describing synovial sarcoma and SYT-SSX1 orSSX2 translocation. Also see Kawai, et al, N. Engl. J. Med338(3):153-160 (1998); Noguchi, et al. int. J. Cancer 72(6):995-1002(1997), Hibshoosh, et al., Semin. Oncol 24(5):515-525 (1997), Shipley,et al., Am. J. Pathol. 148(2):559-567 (1996); Fligman, et al. Am. J.Pathol. 147(6); 1592-1599 (1995). Also see Chand, et al., Genomics30(3):545-552 (1995), Brett, et al., Hum. Mol Genet 6(9): 1559-1564(1997), deBruyn, et al, Oncogene (13/3):643-648. The SSX3 gene isdescribed by deLeeuw, et al, Cytogenet Cell Genet 73(3):179-1983 (1966).

Application of a modification of the SEREX technology described suprahas been used, together with other techniques, to clone two, additionalSSX genes, referred to as SSX4 and SSX5 hereafter as well as analternate splice variant of the SSX4 gene. Specifically, while the SEREXmethodology utilizes autologous serum, the methods set forth infra useallogenic serum.

Motif analysis is a tool which permits one to ascertain what regions ofa longer protein may in fact be of particular interest as binders of MHCor HLA molecules. Essentially, one works with an amino acid motif, whichgenerally includes at least two, and sometimes more, defined amino acidsin a sequence of 8-12 amino acids. This motif is then used to screen alonger sequence to determine which sequences within the longer sequenceconstitute peptides which would bind to an HLA or MHC molecule, andpossibly stimulate proliferation of cytolytic T lymphocytes withspecificity to complexes of the peptide and MHC/HLA molecule. Motifsdiffer for different MHC/HLA molecules. Much work has been done in thisarea, but it is ongoing. As will be seen in the disclosure whichfollows, the inventors have used motif analysis to identify peptideswhich bind to HLA molecules, HLA-A2 molecules in particular.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a peptide titration experiment showing recognition by theCTL of COS-A201 cells pulsed with increasing amounts of the SEQ ID NO:19. The four lines represent separate assays using 4 different CTL linesderived from a stimulation culture.

FIG. 2 depicts the results of cytotoxicity assays to determine HLAspecificity of SEQ ID NO: 19.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

One embodiment of the invention is a novel immunogenic peptide, SEQ IDNO: 19 (Arg-Leu-Leu-Glu-Phe-Tyr-Leu-Ala-Met), that is capable ofstimulating proliferation of cytolytic T cells and inducing a cellularimmune response against cells with SEQ ID NO: 19 complex on the cellsurface.

SEQ ID NO: 19 may be used as a vaccine either therapeutically orprophylactically. It may be administered, for example, to a patientsuffering from a disorder to stimulate one or more components of thepatient's immune system, such as cytotoxic T lymphocytes, to mount acellular immune response against the cells presenting the complex. Thedisorder may be a neoplasia, such as a melanoma. SEQ ID NO: 19 may beadministered directly. Preferably, SEQ ID NO: 19 is administered as acomplex with an HLA molecule or an HLA molecule fragment to stimulateHLA specific cytotoxic lymphocyte reaction.

In prophylactic usage, SEQ ID NO: 19 or SEQ ID NO: 19 conjugated to aHLA molecule is provided to a patient who is in a high risk group fordeveloping a disorder such as a neoplastic disorder. High risk groupsmay include patients with a family history of neoplastic disorders,patients with a genetic predisposition to certain neoplastic disorders,and patients with habits and lifestyles which predispose them to a highrisk of such disorders. Examples of high risk groups include, forexample, Xeroderma Pigmentosum patients who have an increased risk formelanoma, cigarette smokers who have an increased risk for small celllung carcinoma, and Beckwith-Wiedemann syndrome patients who have anincreased risk for hepatoblastoma, and adrenal carcinoma. Inprophylactic usage, SEQ ID NO: 19 by itself or complexed with an HLAmolecule is administered in advance of any indications of neoplasticdisorder to stimulate the patient's immune system to prevent neoplasticdisorder or to attenuate any undiscovered neoplastic disorder.Prophylactic usage also includes the administration of SEQ ID NO: 19 toa patient recovering from a neoplastic disorder treatment such as a bonemarrow transplant or tumor excision to prevent re-emergence of theneoplasia.

In a preferred embodiment, mammals, including humans, who are at highrisk for a neoplastic disorder are treated with vaccines comprising SEQID NO: 19 or SEQ ID NO: 19 complexed with HLA. The vaccine may be in theform of a virus which can infect a cell and induce presentation of SEQID NO: 19 on the cell surface. Alternatively, the vaccine may be anattenuated bacterium, which expresses SEQ ID NO: 19 and a HLA moleculeon its cell surface. In one embodiment, the vaccine may be a mammaliancell transfected by a nucleic acid molecule that encodes SEQ ID NO: 19.

Another embodiment of the invention is directed to the use of SEQ ID NO:19 to generate antisera for prophylactic or treatment purposes. Antiseramay be produced using methods known in the art. For example, SEQ ID NO:19 by itself or in complex with HLA molecules may be injected intoanimals. Antibody titer to SEQ ID NO: 19 may be monitored by withdrawingblood from the animals at regular intervals and analyzing serum titer.Booster shots of additional SEQ ID NO: 19 or SEQ ID NO: 19/HLA complexesto stimulate antibody production may be administered if needed.Alternatively, a blood sample from a patient treated with SEQ ID NO: 19either therapeutically or prophylactically may be used to prepareantisera.

In addition, monoclonal antibodies to SEQ ID NO: 19 may be made usingconventional monoclonal antibody techniques. Further, to improve theefficacy of the monoclonal antibody, it may be humanized. In analternative embodiment, antibodies or antisera may be made in a nonhumanmammal and used. It is understood that all antibodies and antisera maybe purified by antigen immunoaffinity column. In antigen immunoaffinitypurification, the antigen which is immobilized on the solid phase may beSEQ ID NO: 19 alone or SEQ ID NO: 19 complexed with a HLA molecule orHLA molecule fragment. Preferred HLA molecules include HLA-A2 moleculessuch as HLA-A*0201.

The antibodies and antisera produced may be conjugated to toxicmolecules or a detectable label and used to target neoplastic cells fordestruction or detection. A toxin may include, for example, ricin.Detectable labels may include, for example, radioisotopes ³²P, ¹²⁵I, and¹¹¹In. It is understood that some molecules, such as ¹³¹ I, can be botha detectable label and a toxin.

Another embodiment of the invention is directed to a method ofdiagnosing a neoplastic disorder using an antibody that specificallybinds SEQ ID NO: 19. For example, a SEQ ID NO: 19 specific antibody maybe coupled to a detectable label and used in immunocytochemistry todetermine the presence of neoplastic disorder in a biopsy, in an in-situhybridization, and in fluorescent activator sorting.

In another embodiment of the invention, the antibody to SEQ ID NO: 19may be used to purify SEQ ID NO: 19 by immunoaffinity chromatography.Protocols for antibody purification and antibody affinity purificationare commonly known. Chromatography media for antibody purification andaffinity purification are available commercially (e.g., PharmaciaBiotech, Uppsala, Sweden) and detailed protocols for performing thesetechniques are supplied with the media.

In another embodiment of the invention, the antibody to SEQ ID NO: 19may be used as a fluorescent activated cell sorter marker during bonemarrow sorting. For example, in an autologous bone marrow transplant asa treatment for cancer, a patient's extracted bone marrow cells aresorted to separate the neoplastic cells from normal cells. Theneoplastic cells are discarded while the normal cells are reintroducedinto a patient after the patient has undergone cancer treatment. Thesorting (or multiple sortings) is performed with a fluorescent activatedcell sorter after the cells are contacted with fluorescent labeledneoplastic cell specific antibody. The antibodies and antisera of theinvention, specific for SEQ ID NO: 19, may be fluorescent labeled andused as the fluorescent antibody for cell sorting.

Another embodiment of the invention is directed to a method of enhancingstem cell transplantation using SEQ ID NO: 19. Stem cell transplantationis used in the treatment of neoplastic disorder where the treatment(i.e., chemotherapy) destroys the stem cells of a patient. Without astem cell transplant, the patient may die. In a stem celltransplantation, stem cells (marrow cells) are isolated from ahistocompatible donor and injected into a host. The transplanted stemcells, obtained from the marrow, generally contain a fair portion of Tlymphocytes. The T lymphocytes do not affect the stem cell transplantand are not removed prior to transplantation. Thus, T lymphocytes are apart of most stem cell transplants. If the T lymphocytes are properlystimulated, they may initiate an immune response against any possibleresidual neoplastic cells in the host. In the method, the donor stemcell population comprising T lymphocytes is treated with SEQ ID NO: 19or SEQ ID NO: 19 complexed to an HLA peptide before it is deposited intothe patient. The treatment may stimulate the immune response of the Tlymphocyte in the donor stem cell.

Another embodiment of the invention is a novel, substantially purifiedand isolated nucleic acid molecule encoding a peptide with an amino acidsequence of SEQ ID NO: 19 (Arg-Leu-Leu-Glu-Phe-Tyr-Leu-Ala-Met). Thenucleic acid that encodes SEQ ID NO: 19 may be deduced from the aminoacid sequence of SEQ ID NO: 19. It is known that the nucleic acid codeis degenerate. In this case, for example, Arginine is encoded by thecodons, CGT, CGC, CGA, CGG, AGA and AGG. Leucine is encoded by thecodons TTA, TTG, CTT, CTC, CTA and CTG. Glutamic Acid is encoded by thecodons GAA or GAG. Phe is encoded by the codons TTT and TTC. Tyr isencoded by the codons TAT and TAC. Alanine is encoded by the codons GCT,GCC, GCA, and GCG. Met is encoded by the codon ATG. Thus, the nucleicacid molecule may have any sequence which encodes a peptide of SEQ IDNO: 19. Preferably, the nucleic acid is in operable linkage with apromoter which can express SEQ ID NO: 19 constitutively or uponinduction in a eukaryotic or prokaryotic host. More preferably, theplasmid may have a wide host range allowing replication multiple hostssuch as yeast and bacteria.

Another embodiment of the invention is directed to novel pharmaceuticalcompositions useful for treating a neoplastic disorder. Thepharmaceutical composition contains between 0.001% to 100% by weight ofSEQ ID NO: 19 in a pharmaceutically acceptable carrier and/or diluent.Suitable carriers may be bovine serum albumin and suitable diluents maybe phosphate buffered saline or distilled water. In a preferredembodiment, SEQ ID NO: 19 is bound to an HLA molecule for stimulation ofthe recipient's immune response. Preferred HLA molecules include HLA-A2molecules such as HLA-A*0201.

Another embodiment of the invention is a method for ameliorating thesymptoms of a neoplastic disorder by administering a pharmaceuticalcomposition comprising SEQ ID NO: 19 to a patient. Administration may beperformed by means known in the art including topical administration,injection, intravenous drip, implantation of a slow release device,topical administration and aerosol administration. The therapeuticeffects of SEQ ID NO: 19 may be enhanced by the addition of an adjuvant,a T cell booster, or a cytokine. Adjuvants and cytokines that canenhance T lymphocyte response to antigens are known and include, forexample, microorganisms such as BCG, T cell boosters such as lentinan,and cytokines such as IL-1, IL-2, IL-4, IL-6, IL-12 and TNF.

Another embodiment of the invention is directed to a host cell or viruscontaining a DNA molecule which encodes the peptide of the invention.The host cell or virus may be used in a method to produce the peptide.Further, the host cell or virus may be used as a live, or attenuatedvaccine to vaccinate against a neoplastic disorder.

Another embodiment of the invention is directed to a method to provide avaccine for preventing a neoplastic disorder. The vaccine may be a virusor bacterium containing a DNA molecule that encodes a peptide of theinvention.

Another embodiment of the invention is directed to a method of making apeptide of the invention by culturing a virus or bacteria containing aDNA molecule that encodes a peptide of the invention. Methods forpeptide expression are known. For example, the peptide may be producedby transfecting an expression vector containing a nucleic acid sequencethat encodes SEQ ID NO: 19 into a host cell and inducing expression. Thehost cell may be a prokaryotic (bacterium), yeast, insect, or mammaliancell. Various methods of host cell expression are well known. Reagents,vectors, cell lines and detailed expression protocols are commerciallyavailable (e.g., Invitrogen (Carlsbad, Calif.), Stratagene (La Jolla,Calif.)). For example, SEQ ID NO: 19 may be expressed as athioredoxin-SEQ ID NO: 19 fusion protein under the ThioFusion™Expression system of Invitrogen. Following expression, the fusionpeptide may be purified by a metal binding resin with specific affinityfor thioredoxin part of the fusion protein. The fusion protein, stillbound to the metal binding resin, may be cleaved by enterokinase tospecifically release intact SEQ ID NO: 19. Alternatively, SEQ ID NO: 19may be synthesized and purified by commercially available peptidesynthesis machines (e.g., PE Corporation (Norwalk, Conn.); AdvancedChemTech (Louisville, Ky.)).

Another embodiment of the invention is directed to a polytope moleculecomprising a plurality of sequences corresponding to amino acidsequences which bind to MHC molecules, at least one of which is theamino acid sequence of SEQ ID NO: 19. A polytope is a polypeptidecomprising two or more MHC binding sequence. A nucleic acid moleculeencoding a polytope may be made by ligating together oligonucleotidesencoding different peptides. The polytope encoding nucleic acid moleculemay be placed in an expression vector in functional linkage with apromoter for expressing a recombinant polytope in a host cell. Therecombinant polytope polypeptide of the invention may comprise from 2 to1000, preferably 4 to 200 and most preferably between 4 and 20 peptides;wherein at least one of them is SEQ ID NO: 19. In addition, the peptidesmay be sandwiched between recognition sites for a sequence specificcleavage enzyme. Sequence specific cleavage enzymes may be, for example,enterokinase or thrombin which recognizes and cleaves at knownsequences. Thus, cleavage of the polytope will result in multiplepeptides which can bind to a MHC molecule.

Another embodiment of the invention is directed to a method of treatinga patient with neoplastic disorder with SEQ ID NO: 19. In the method, Tlymphocytes isolated from a patient are treated with SEQ ID NO: 19 tosensitize the T cells to SEQ ID NO: 19. The treated T lymphocytes arereintroduced into the patient to stimulate the patient's response toneoplastic cells. Optionally, the treated T lymphocytes may be culturedand amplified before reintroduction to enhance the effects of SEQ ID NO:19.

Another embodiment of the invention is directed to a composition ofmatter comprising a peptide of SEQ ID NO: 19. The composition of mattermay include, in addition to SEQ ID NO: 19, one or more disorderassociated antigens, HLA molecules or HLA molecule fragments. Thecompositions may be tailored to elicit a CTL reaction against specificcells or cell populations when the composition is administered to apatient. For example, the composition may comprise multiple cell surfacemarkers expressed by a targeted cell population. The cell surfacemarkers may include, for example, HLA antigens and disorder associatedantigens.

EXAMPLE 1

A human testicular cDNA expression library was obtained, and screened,with serum from a melanoma patient identified as MZ2. See e.g., parentapplication U.S. patent application Ser. No. 08/479,328 incorporated byreference; also see U.S. patent application Ser. No. 08/725,182 alsoincorporated by reference; Sahin, et al., Proc. Natl. Acad. Sci. USA92:11810-11813 (1995). This serum had been treated using the methodologydescribed in these references. Briefly, serum was diluted 1:10, and thenpreabsorbed with transfected E. coli lysate. Following thispreabsorption step, the absorbed serum was diluted 1:10, for a finaldilution of 1:100. Following the final dilution the samples wereincubated overnight at room temperature, with nitrocellulose membranescontaining phage plaques prepared using the methodology referred tosupra. The nitrocellulose membranes were washed, incubated with alkalinephosphatase conjugated goat anti-human Fc_(γ) secondary antibodies, andthe reaction was observed with the substrates 5-bromo-4-chloro-3-indolylphosphate and nitroblue tetrazolium. In a secondary screen, anyphagemids which encoded human immunoglobulin were eliminated.

A total of 3.6×10⁵ pfus were screened, resulting in eight positiveclones. Standard sequencing reactions were carried out, and thesequences were compared to sequence banks of known sequences.

Of the eight clones, two were found to code for known autoimmune diseaseassociated molecules, i.e., Golgin-95 (Fritzler, et al., J. Exp.Med.178:49-62 (1993)), and human upstream binding factor (Chan, et al.,J. Exp. Med. 174:1239-1244 (1991)). Three other clones were found toencode for proteins which are widely expressed in human tissue, i.e.,ribosomal receptor, collagen type VI globular domain, and rapamycinbinding protein. Of the remaining three sequences, one was found to benon-homologous to any known sequence, but was expressed ubiquitously inhuman tissues (this was found via RT-PCR analysis, but details are notprovided herein). The remaining two were found to be identical to fulllength HOM-MEL-40, described in Ser. No. 08/479,328, while the eighthclone was found to be almost identical to “SSX3,” as described byDeLeeuw, et al., Cytogenet. Cell Genet 73:179-183 (1996), differingtherefrom in only two base pair differences in the coding region. Thesedifferences are probably artifactual in nature; however, the clone alsoincluded a 43 base pair 3′-untranslated region.

EXAMPLE 2

In order to carry out Southern blotting experiments, described infra,the SSX genes were amplified, using RT-PCR.

To do this, two primers were prepared using the published SSX2 sequencei.e., MEL-40A:

5′-CACACAGGAT CCATGAACGG AGA  (SEQ ID NO: 3),

and

MEL-40B:

5′-CACACAAAGC TTTGAGGGGA GTTACTCGTC ATC  (SEQ. ID NO: 4)

See Crew, et al., EMBO J 14:2333-2340 (1995). Amplification was thencarried out using 0.25 U Taq polymerase in a 25 μl reaction volume,using an annealing temperature of 60° C. A total of 35 cycles werecarried out.

EXAMPLE 3

The RT-PCR methodology described supra was carried out on testiculartotal RNA, and the amplification product was used in southern blottingexperiments.

Genomic DNA was extracted from non-neoplastic tissue samples, and thensubjected to restriction enzyme digestion, using BamHI, Eco RI, orHindIII in separate experiments and then separated on a 0.7% agarosegel, followed by blotting onto nitrocellulose filters. The amplificationproducts described supra were labeled with ³²P, using well-knownmethods, and the labeled materials were then used as probes under highstringency conditions (65 ° C., aqueous buffer), followed by highstringency washes, ending with a final wash at 0.2×SSC, 0.2% SDS, 65° C.

The Southern blotting revealed more than 10 bands, in each case (i.e.,each of the BamHI, EcoRI, and HindIII digests), strongly suggesting thatthere is a family of SSX genes which contained more than the threeidentified previously. In view of this observation, an approach wasdesigned which combined both PCR cloning, and restriction map analysis,to identify other SSX genes.

EXAMPLE 4

When the sequences of SSX1, 2 and 3 were compared, it was found thatthey shared highly conserved 5′ and 3′ regions, which explained why theolignucleotides of SEQ ID NOS: 3 and 4 were capable of amplifying allthree sequences under the recited conditions, and suggested that thishomology was shared by the family of SSX genes, whatever its size.Hence, the oligonucleotides of SEQ ID NOS: 3 and 4 would be sufficientto amplify the other members of the SSX gene family.

An analysis of the sequences of SSX1, 2 and 3 revealed that SSXI and 2contained a BglII site which was not shared by SSX3. Similarly, SSX3contained an EcoRV site not shared by the other genes.

In view of this information, testicular cDNA was amplified, using SEQ IDNOS: 3 and 4, as described supra, and was then subjected to BglIIdigestion. Any BglII resistant sequences were then cloned, sequenced,and compared with the known sequences.

This resulted in the identification of two previously unidentifiedsequences, referred to hereafter as SSX4 and SSX5, presented as SEQ IDNOS: 5 and 6 herein. A search of the GenBank database found two clones,identified by Accession Number N24445 and W00507, both of whichconsisted of a sequence-tag-derived cDNA segment. The clone identifiedby N24445 contained the 3′-untranslated region of SSX4, and part of itscoding sequence, while the one identified as W00507 contained a shorterfragment of the 3′-untranslated region of SSX4, and a longer part of thecoding sequence. Specifically, N24445 consists of base 344 of SSX4 (SEQID NO:5), through the 3-end, plus 319 bases 3′ of the stop codon. TheW00507 sequence consists of a 99 base pair sequence, showing no homologyto SSX genes followed by a region identical to nucleotides 280 throughthe end of SEQ ID NO:5, through 67 bases 3′ of the stop codon of SEQ IDNO: 1.

Two forms of SSX4 (SEQ ID NO: 5) were identified. One of these lackednucleotides 331 to 466 but was otherwise identical to SSX4 as presentedin SEQ ID NO: 5. As is described infra, the shorter form is analternatively spliced variant.

In Table 1, which follows, the nucleotide and amino acid sequences ofthe 5 known members of the SSX family are compared. One reads the tablehorizontally for nucleotide homology, and vertically for amino acidhomology.

TABLE 1 Nucleotide and amino acid homology among SSX family membersNucleotide Sequence Homology (%) SSX1 SSX2 SSX3 SSX4 SSX5 SSX1 89.1 89.689.4 88.7 SSX2 78.2 95.1 91.5 92.9 SSX3 77.7 91.0 91.1 92.7 SSX4 79.379.8 80.9 89.8 SSX5 76.6 83.5 84.0 77.7 Amino Acid Sequence Homology (%)

Hence, SSX1 and SSX4 share 89.4% homology on the nucleotide level, and79.3% homology on the amino acid level.

When the truncated form of SSX4 is analyzed, it has an amino acidsequence completely different from others, due to alternate splicing andshifting of a downstream open reading frame. The putative protein is 153amino acids long, and the 42 carboxy terminal amino acids show nohomology to the other SSX proteins.

EXAMPLE 5

The genomic organization of the SSX2 genes was then studied. To do this,a genomic human placental library (in lambda phage) was screened, usingthe same protocol and probes described supra in the discussion of thesouthern blotting work. Any positive primary clones were purified, viatwo additional rounds of cloning.

Multiple positive clones were isolated, one of which was partiallysequenced, and identified as the genomic clone of SSX2. A series ofexperiments carrying out standard subcloning and sequencing workfollowed, so as to define the exon—intron boundaries.

The analysis revealed that the SSX2, gene contains six exons, and spansat least 8 kilobases. All defined boundaries were found to observe theconsensus sequence of exon/intron junctions, i.e. GT/AG.

The alternate splice variant of SSX4, discussed supra, was found to lackthe fifth exon in the coding region. This was ascertained by comparingit to the SSX2 genomic clone, and drawing correlations therefrom.

EXAMPLE 6

The expression of individual SSX genes in normal and tumor tissues wasthen examined. This required the construction of specific primers, basedupon the known sequences, and these follow, as SEQ ID NOS: 7-16:

TABLE 2 Gene-specific PCR primer sequences for individual SSX genes SSX1A (5′): 5′-CTAAAGCATCAGAGAAGAGAAGC [nt.44-66] SEQ ID NO: 7 SSX 1B (3′):5′-AGATCTCTTATTAATCTTCTCAGAAA [nt.440-65] SEQ ID NO: 8 SSX 2A (5′):5′-GTGCTCAAATACCAGAGAAGATC [nt.41-63] SEQ ID NO: 9 SSX 2B (3′):5′-TTTTGGGTCCAGATCTCTCGTG [nt.102-25] SEQ ID NO: 10 SSX 3A (5′):5′-GGAAGAGTGGGAAAAGATGAAAGT [nt.454-75] SEQ ID NO: 11 SSX 3B (3′):5′-CCCCTTTTGGGTCCAGATATCA [nt.458-79] SEQ ID NO: 12 SSX 4A (5′):5′-AAATCGTCTATGTGTATATGAAGCT [nt.133-58] SEQ ID NO: 13 SSX 4B (3′):5′-GGGTCGCTGATCTCTTCATAAAC [nt.526-48] SEQ ID NO: 14 SSX 5A (5′):5′-GTTCTCAAATACCACAGAAGATG [nt.39-63] SEQ ID NO: 15 SSX 5B (3′):5′-CTCTGCTGGCTTCTCGGGCCG [nt.335-54] SEQ ID NO: 16

The specificity of the clones was confirmed by amplifying the previouslyidentified cDNA for SSX1 through SSX5. Taq polymerase was used, at 60°C. for SSX1 and 4, and 65° C. for SSX2, 3 and 5. Each set of primerpairs was found to be specific, except that the SSX2 primers were foundto amplify minute (less than 1/20 of SSX2) amounts of SSX3 plasmid DNA.

Once the specificity was confirmed, the primers were used to analyzetesticular mRNA, using the RT-PCR protocols set forth supra.

The expected PCR products were found in all 5 cases, and amplificationwith the SSX4 pair did result in two amplification products, which isconsistent with alternative splice variants.

The expression of SSX genes in cultured melanocytes was then studied.RT-PCR was carried out, using the protocols set forth supra. No PCRproduct was found. Reamplification resulted in a small amount of SSX4product, including both alternate forms, indicating that SSX4 expressionin cultured melanocytes is inconsistent and is at very low levels whenit occurs.

This analysis was then extended to a panel of twelve melanoma celllines. These results are set forth in the following table.

TABLE 3 SSX expression in melanoma cell lines detected by RT-PCR* SSX1SSX2 SSX3 SSX4 SSX5 MZ2-Mel 2.2 + + − − − MZ2-Mel 3.1 + + − − −SK-MEL-13 − − − − − SK-MEL-19 − − − − − SK-MEL-23 − − − − − SK-MEL-29 −− − − − SK-MEL-30  −*  −* −  −* − SK-MEL-31 − − − − − SK-MEL-33 − − − −− SK-MEL-37 + + − + + SK-MEL-179 − − − − − M24-MET − − − − − *Positive(+) denotes strong expression. Weak positivity was observedinconsistently in SK-MEL-30 for SSX 1,2, and 4, likely representing lowlevel expression.

EXAMPLE 7

Additional experiments were carried out to analyze expression of themembers of the SSX family in various tumors. To do this, total cellularRNA was extracted from frozen tissue specimens using guanidiumisothiocyanate for denaturation followed by acidic phenol extraction andisopropanol precipitation, as described by Chomczynski, et al, Ann.Biochem 162: 156-159 (1987), incorporated by reference. Samples of totalRNA (4 ug) were primed with oligoDT(18) primers, and reversetranscribed, following standard methodologies. The integrity of the cDNAthus obtained was tested via amplifying B-acin transcripts in a 25cycle, standard PCR, as described by Tureci, et al, Canc. Res. 56:4766-4772 (1996).

In order to carry out PCR analyses, the primers listed as SEQ ID NOS:5-14, supra were used, as well as SEQ ID NOS: 17 and 18, i.e.:

ACAGCATTAC CAAGGACAGC AGCCACC SEQ ID NO: 17 GCCAACAGCA AGATGCATACCAGGGAC SEQ ID NO: 18

These two sequences were each used with both SEQ ID NOS: 6 and 8 inorder to detect the SYT/SSX fusion transcript reported for synovialsarcoma by Clark et al, supra, and Crew, et al, supra. The amplificationwas carried out by amplifying 1 μl of first strand cDNA with 10 pMol ofeach dNTP, and 1.67 mN MgCl₂ in a 30 μl reaction. Following 12 minutesat 94° C. to activate the enzyme, 35 cycles of PCR were performed. Eachcycle consisted of 1 minute for annealing (56° C. for SEQ ID NOS: 7 & 8;67° C. for SEQ ID NOS: 9 & 10; 65° C. for SEQ ID NOS: 11 & 12; 60° C.for SEQ ID NOS: 13 & 14; 66° C. for SEQ ID NOS: 15 & 16; 60° C. for SEQID NOS: 17 & 8 and 18 & 10), followed by 2 minutes at 72° C., 1 minuteat 94° C., and a final elongation step at 72° C. for 8 minutes. A 15 μlaliquot of each reaction was size fractionated on a 2% agarose gel,visualized with ethidium bromide staining, and assessed for expectedsize. The expected sizes were 421 base pairs for SEQ ID NOS: 7 & 8; 435base pairs for SEQ ID NOS: 9 & 10; 381 base pairs for SEQ ID NOS 11 &12; 413 base pairs for SEQ ID NOS: 13 & 14, and 324 base pairs for SEQID NOS: 15 & 16. The conditions chosen were stringent, so as to preventcross anneling of primers to other members of the SSX family. Additionalsteps were also taken to ensure that the RT-PCR products were derivedfrom cDNA, and not contaminating DNA. Each experiment was done intriplicate. A total of 325 tumor specimens were analyzed. The resultsare presented in Tables 4 & 5 which follow.

It is to be noted that while most of the SSX positive tumors expressedonly one member of the SSX family, several tumor types showedcoexpression of two or more genes.

Expression of SSX genes in synovial sarcoma was analyzed, because theliterature reports that all synovial sarcoma cases analyzed have beenshown to carry either the SYT/SSX1 or SYT/SSX2 translocation, atbreakpoints flanked by the primer sets discussed herein, i.e., SEQ IDNO: 17/SEQ ID NO: 8; SEQ ID NO: 17/SEQ ID NO: 10; SEQ ID NO. 17/SEQ IDNO: 8; SEQ ID NO: 18/SEQ ID NO: 10. The PCR work described supra showedthat SYT/SSX1 translocations were found in three of the synovial sarcomasamples tested, while SYT/SSX2 was found in one. The one in which it wasfound was also one in which SYT/SSX1 was found. Expression of SSXappeared to be independent of translocation.

TABLE 4 Expression of SSX genes by human neoplasms at lease Tissues oneTumor entity tested SSX1 SSX2 SSX3 SSX4 SSX5 positive % Lymphoma 11 —  4— — —  4 36 Breast cancer 67  5  5 — 10 — 16 23 Endometrial cancer  8  1 2 —  1 1  1 13 Colorectal cancer 58  3  7 —  9 1 16 27 Ovarian cancer12 — — —  6 —  6 50 Renal cell cancer 22 —  1 — — —  1  4 Malignantmelanoma 37 10 13 — 10 2 16 43 Glioma 31 —  2 —  3 —  5 16 Lung cancer24  1  4 —  1 1  5 21 Stomach cancer  3 — — —  1 —  1 33 Prostaticcancer  5 —  2 — — —  2 40 Bladder cancer  9  2  4 —  2 —  5 55Head-Neck cancer 14  3  5 —  4 1  8 57 Synovial sarcoma  4 —  2 —  1 1 3 75 Leukemia 23 — — — — —  0  0 Leiomyosarcoma  6 — — — — —  0  0Thyroid cancer  4 — — — — —  0  0 Seminoma  2 — — — — —  0  0 Total 325 25 50 0 48 7 89

TABLE 5 Expression pattern of individual SSX genes in SSX-positive tumorsamples.¹ Breast Cancer (67 specimens) SSX1 SSX2 SSX4 SSX5 51 specimens− − − −  7 specimens − − + −  4 specimens − + − −  2 specimens + − − − 2 specimens + − + −  1 specimen + + + − Melanoma (37 specimens) SSX1SSX2 SSX4 SSX5 21 specimens − − − −  5 specimens + + + −  4 specimens− + − −  2 specimens − + + −  1 specimen + − − −  1 specimen + + − −  1specimen + − + −  1 specimen + − + +  1 specimen + + + + Endomet. Cancer(8 specimens) SSX1 SSX2 SSX4 SSX5  7 specimens − − − −  1specimen + + + + Glioma (31 specimens) SSX1 SSX2 SSX4 SSX5 25 specimens− − − −  3 specimens − + − −  2 specimens − − + − Lung Cancer (24specimens) SSX1 SSX2 SSX4 SSX5 19 specimens − − − −  3 specimens − + − − 1 specimen − − − +  1 specimen + + + − Colorectal Cancer (58 specimens)SSX1 SSX2 SSX4 SSX5 42 specimens − − − −  7 specimens − + − −  5specimens − − + −  3 specimens + − + −  1 specimen − − + + BladderCancer (9 specimens) SSX1 SSX2 SSX4 SSX5  4 specimens − − − −  2specimens − + − −  1 specimen − − + −  1 specimen + + − −  1specimen + + + − Head-Neck Cancer (14 specimens) SSX1 SSX2 SSX4 SSX5  6specimens − − − −  2 specimens + − − −  2 specimens − + + −  1 specimen− + − −  1 specimen − − + −  1 specimen + + − −  1 specimen − + + +Synovial Sarcoma (4 specimens) SSX1 SSX2 SSX4 SSX5 SYT/SSX1 SYT/SSX5 Sy1− − + − + − Sy2 − + − + + − Sy3 − − − − − + Sy4 − + − − + −

EXAMPLE 8

This example details further experiments designed to identify additionalpeptides which bind to HLA-A2 molecules, and which stimulate CTLproliferation.

First, peripheral blood mononuclear cells (“PBMCs” hereafter) wereisolated from the blood of healthy HLA-A*0201⁺ donors, using standardFicoll-Hypaque methods. These PBMCs were then treated to separateadherent monocytes from non-adherent peripheral blood lymphocytes(“PBLs”), by incubating the cells for 1-2 hours, at 37° C., on plasticsurfaces. Any non-adherent PBLs were cryopreserved until needed infurther experiments. The adherent cells were stimulated to differentiateinto dendritic cells by incubating them in AIMV medium supplemented with1000 U/ml of IL-4, and 1000 U/ml of GM-CSF. The cells were incubated for5 days.

Seven days after incubation began, samples of the dendritic cells(8×10⁵) were loaded with 50 μg/ml of exogenously added peptide. (Detailsof the peptides are provided infra). Loading continued for 2 hours, at37° C., in a medium which contained 1000 U/ml of TNF-α and 10,000 U/mlIL-1β. The peptide pulsed dendritic cells were then washed, twice, inexcess, peptide free medium. Autologous PBLs, obtained as described,supra, were thawed, and 4×10⁷ PBLs were then combined with 8×10⁵ peptideleaded dendritic cells, (ratio: 50:1), in a medium which contained 5ng/ml of IL-7 and 20 U/ml of IL-2. The cultures were then incubated at37° C.

Lymphocyte cultures were restimulated at 14, 21, and 28 days, in thesame manner as the experiment carried out after 7 days. Cytotoxicityassays were carried out, at 14, 21, and 28 days, using a europiumrelease assay, as described by Blomberg, et al., J. Immunol. Meth. 114:191-195 (1988), incorporated by reference, or the commercially availableELISPOT assay, which measures IFN-γ release.

The peptides which were tested were all derived from the amino acidsequence of NY-ESO-1 as is described in U.S. Pat. No. 5,804,381, toChen, et al., incorporated by reference, or the amino acid sequences ofSSX-4. The peptides tested were:

RLLEFYLAM  (SEQ ID NO: 19)

and

SLAQDAPPL  (SEQ ID NO: 20)

both of which are derived from NY-ESO-1, and

STLEKINKT  (SEQ ID NO: 21)

derived from SSX-4. The two NY-ESO-1 derived peptides were tested inELISPOT assays. The results follow. In summary, three experiments werecarried out. The results are presented in terms of the number of spots(positives) secured when the HLA-A2 positive cells were pulsed with thepeptide minus the number of spots obtained using non-pulsed cells. Asindicated, measurements were taken at 14, 21 and 28 days.

The following results are for peptide RLLEFYLAM (SEQ ID NO.19).

Day Measured (Pulsed Cells - Unpulsed Cells) 14 21 28 Expt 1 30 8 * Expt2 22 * 12 Expt 3  6 * 12 *not determined

EXAMPLE 9

In follow up experiments, the T cell cultures described supra weretested on both COS cells which had been transfected with HLA-A*0201encoding cDNA and were pulsed with endogenous peptide, as describedsupra, or COS cells which had been transfected with both HLA-A*0201 andNY-ESO-1 encoding sequences. Again, the ELISPOT assay was used, for bothtypes of COS transfectants. Six different cultures of T cells weretested, in two experiments per culture.

Pulsed with Endogenous Peptide NY-ESO-1 Production Culture 1 Expt 1 6444 Expt 2 44 52 Culture 2 Expt 1 48 45 Expt 2 100 64 Culture 3 Expt 1 2037 Expt 2 16 16 Culture 4 Expt 1 17 40 Expt 2 28 34 Culture 5 Expt 1 3626 Expt 2 4 36 Culture 6 Expt 1 12 62 Expt 2 44 96

The fact that the endogenous NY-ESO-1 led to lysis suggests thatNY-ESO-1 is processed to this peptide via HLA-A2 positive cells.

Similar experiments were carried out with the second NY-ESO-1 derivedpeptide, i.e., SLAQDAPPL (SEQ ID NO. 20). These results follow:

Pulsed with Endogenous Peptide NY-ESO-1 Production Culture 1 Expt 1 2816 Expt 2 30 14 Culture 2 Expt 1 31 75 Expt 2 30 70 Culture 3 Expt 1 3244

EXAMPLE 10

In further experiments, the specificity of the CTLs generated in theprior experiment was tested by combining these CTLs with COS cells,transfected with HLA-A*0201 encoding sequences, which were then pulsedwith peptide. First, the peptide RLLEFYLAM (SEQ ID NO. 19) was tested,in three experiments, and then SLAQDAPPL (SEQ ID NO. 20) was tested, insix experiments. Europium release was measured, as described supra, andthe percent of target cells lysed was determined. The results follow:

% LYSIS Peptide Added No Peptide PEPTIDE RLLEFYLAM (SEQ ID NO. 19) Expt1 43 0 Expt 2 8 0 Expt 3 9 0 PEPTIDE SLAQDAPPL (SEQ ID NO. 20) Expt 1 110 Expt 2 13 0 Expt 3 13 0 Expt 4 21 0 Expt 5 12 0 Expt 6 42 0

In additional experiments, the CTLs specific to RLLEFYLAM (SEQ ID NO.19)/HLA-A2 complexes also recognized and lysed melanoma cell lineSK-Mel-37 which is known to express both HLA-A2 and NY-ESO-1. Thisrecognition was inhibited via preincubating the target cells with anHLA-A2 binding monoclonal antibody, BB7.2. This confirmed that the CTLswere HLA-A2 specific for the complexes of the peptide and HLA-A2.

In a second set of experiments, a peptide titration experiment wasperformed to further determine the ability of SEQ ID NO: 19 to induce aCTL response. Experiments were conducted substantially in accordancewith the protocol of Example 8. Samples of the cells (8×10⁵) were loadedwith 1 μM, 5 μM, 10 μM and 50 μM of SEQ ID NO: 19 for 2 hours at 37° C.in a medium containing 1000 U/ml of TNF-α. Cytotoxicity assays wereperformed using a europium release assay as described supra. In fourseparate sets of experiments, increasing concentrations of SEQ ID NO: 19cause an increase in lysis. The data is plotted in FIG. 1. The resultsindicate that cells pulsed with SEQ ID NO: 19 can elicit a CTL mediatedlysis.

To determine if the lysis is HLA specific, the CTL assays were performedusing COS cells transfected with a cDNA encoding HLA-A*0201 as describedsupra and pulsed with SEQ ID NO: 19. As shown in FIG. 2, transfected COScells that were not pulsed with peptides had a lysis rate of 5%.HLA-A*0201 transfected COS cells pulsed with SEQ ID NO: 19, showed 55%lysis. This is consistent with the hypothesis that SEQ ID NO: 19, whencomplexed with an HLA molecule, is responsible for increased lysis. Todetermine if the effect was HLA restricted, HLA-A*0201 transfected COScells pulsed with SEQ ID NO: 19 were assayed in the presence ofanti-HLA-A2 antibodies. The lysis decreased to about 10%. This indicatedthat the cytotoxicity is HLA-A2 restricted because the anti-HLA-A2antibody bound to the HLA-A2 molecule, interfering with the recognitionof the HLA-A2/SEQ ID NO: 19 complex by the TCR (T cell receptor) of thespecific T cell, thus inhibiting lysis. Similar experiments using theSK-MEL-37 cell line, which is known to express both NY-ESO-1 and HLA-A2,as the target demonstrated that the peptide was endogenously processedand presented in tumor cells. SK-MEL-37 cells showed about 23% lysiswhereas lysis was reduced to 6% in the presence of anti-HLA-A2, which isconsistent with the findings using COS-A201 cells, described supra.

To determine the cellular location of NY-ESO-1, several cell lines knownto express NY-ESO-1 were stained by immunofluorescence with a NY-ESO-1specific monoclonal antibody (Stockert et al., J. Exp Med 187 1349-54,1998; incorporated herein by reference. Also see U.S. patent applicationSer. No. 09/062,422 filed Apr. 17, 1998, incorporated herein byreference). Upon analysis of the stained cell samples, it was found thatNY-ESO-1 was localized to the endoplasmic reticulum. This is consistentwith the immunogenicity of NY-ESO-1.

EXAMPLE 11

An additional peptide derived from SSX-4, i.e., STLEKINKT (SEQ ID NO:21) was also tested, in the same way the NY-ESO-1 derived peptides weretested. First, ELISPOT assays were carried out, using COS cells whichexpressed HLA-A*0201, and which either expressed full length SSX-4, dueto transfection with cDNA encoding the protein, or which were pulsedwith the peptide. Three cultures were tested, in two experiments. Theresults follow:

Pulsed With Endogenous Peptide NY-ESO-1 Production Culture 1 Expt 1 50100 Expt 2 20 138 Culture 2 Expt 1 8 12 Expt 2 6 14 Culture 3 Expt 1 1547 Expt 2 14 54

Further, as with the NY-ESO-1 peptides, specificity of the CTLs wasconfirmed, using the same assay as described supra, i.e., combining theCTLs generated against the complexes with COS cells, transfected withHLA-A*0201, and pulsed with peptide. The europium release assaydescribed supra was used. The results follow:

% LYSIS Peptide Added No Peptide Expt 1 22 0 Expt 2 14 0 Expt 3 46 0Expt 4 16 0

As with the NY-ESO-1 derived peptides, CTL recognition was inhibited viapreincubation with the monoclonal antibody BB7.2, confirming specificityof the CTL for complexes HLA-A2 and peptides.

EXAMPLE 12

Additional experiments were carried out on peptides derived from SSX-2i.e., KASEKIFYV (SEQ ID NO: 72), and peptides derived from NY-ESO-1,i.e., SLLMWITQCFL (SEQ ID NO: 130), SLLMWITQC (SEQ ID NO: 131), andQLSLLMWIT (SEQ ID NO: 122). In each case, the same type of assays aswere carried out in examples 8-11 were carried out. The results werecomparable, in that for each peptide, CTL were generated which werespecific for the respective peptide/HLA-A2 complex.

EXAMPLE 13

The amino acid sequence of the proteins encoded by the SSX genes wereanalyzed for peptide sequences which correspond to HLA binding motifs.This was done using the algorithm taught by Parker et al., J. Immunol.142: 163 (1994), incorporated by reference, augmented by using, as anadditional motif, nonamers where position 2 is Thr or Ala, and position9 is Thr or Ala. In the information which follows, the amino acidsequence, the HLA molecule to which it presumably binds, and thepositions in the relevant SSX molecule are given. The resultingcomplexes should provoke a cytolytic T cell response. This could bedetermined by one skilled in the art following methods taught by, e.g.,van der Bruggen, et al., J. Eur. J. Immunol. 24: 3038-3043 (1994),incorporated by reference, as well as the protocols set forth inExamples 8-11, supra.

SSX-5 A2 KASEKIIYV 41-49 (SEQ ID NO: 22) DAFVRRPRV  5-13 (SEQ ID NO: 23)QIPQKMQKA 16-24 (SEQ ID NO: 24) MTKLGFKAT 58-66 (SEQ ID NO: 25)MTFGRLQGI  99-107 (SEQ ID NO: 26) NTSEKVNKT 146-154 (SEQ ID NO: 27)YVYMKRKYEA 48-57 (SEQ ID NO:28) YMKRKYEAMT 50-59 (SEQ ID NO: 29)EAMTKLGFKA 56-65 (SEQ ID NO: 30) MTKLGFKATL 58-67 (SEQ ID NO: 31)RLQGIGPKIT 103-112 (SEQ ID NO: 32) QLRPSGKLNT 138-147 (SEQ ID NO: 33) A3GIFPKITPEK 106-115 (SEQ ID NO: 34) KLNTSEKVNK 144-153 (SEQ ID NO: 35)A24 KYEAMTKLGF 54-63 (SEQ ID NO: 36) B7 HPQMTFGRL  96-104 (SEQ ID NO:37) GPQNNGKQL 131-139 (SEQ ID NO: 38) B8 RVRERKQL 167-174 (SEQ ID NO:39) B44 YEAMTKLGF 55-63 (SEQ ID NO: 40) RERKQLVIY 169-177 (SEQ ID NO:41) B52 KQLVIYEEI 172-180 (SEQ ID NO: 42) MTFGRLQGIF  99-108 (SEQ ID NO:43) SSX-4 A2 KSSEKIVYV 41-49 (SEQ ID NO: 44) VMTKLGFKV 57-65 (SEQ ID NO:45) YVYMKLNYEV 48-57 (SEQ ID NO: 46) KLNYEVMTKL 52-61 (SEQ ID NO: 47)FARRPRDDA  7-13 (SEQ ID NO: 48) QISEKLRKA 16-24 (SEQ ID NO: 49)MTFGSLQRI  99-107 (SEQ ID NO: 50) SLQRIFPKI 103-111 (SEQ ID NO: 51)KIVYVYMKL 45-53 (SEQ ID NO: 52) KLRKAFDDI 20-28 (SEQ ID NO: 53)KLRKAFDDIA 20-29 (SEQ ID NO: 54) YMKLNYEVMT 50-59 (SEQ ID NO: 55)MTKLGFKVTL 58-67 (SEQ ID NO: 56) QLCPPGNPST 138-147 (SEQ ID NO: 57) A3KLNYEVMTK 52-60 (SEQ ID NO: 58) NYEVMTKLGF 54-63 (SEQ ID NO: 59) B7RPQMTFGSL  96-104 (SEQ ID NO: 60) KPAEEENGL 115-123 (SEQ ID NO: 61)GPQNDGKQL 131-139 (SEQ ID NO: 62) CPPGNPSTL 140-148 (SEQ ID NO: 63) B8RLRERKQL 167-174 (SEQ ID NO: 64) B35 RPRDDAQI 10-17 (SEQ ID NO: 65)KPAEEENGL 115-123 (SEQ ID NO: 66) B44 YEVMTKLGF 55-63 (SEQ ID NO: 67)RERKQLVVY 169-177 (SEQ ID NO: 68) B52 KQLVVYEEI 172-180 (SEQ ID NO: 69)MTFGSLQRIF  99-108 (SEQ ID NO: 70) SSX-2 A-2 KIQKAFDDI 20-28 (SEQ ID NO:71) KASEKIFYV 41-49 (SEQ ID NO: 72) AMTKLGFKA 57-65 (SEQ ID NO: 73)RLQGISPKI 103-111 (SEQ ID NO: 74) RLRERKQLV 167-175 (SEQ ID NO: 75)DAFARRPTV  5-13 (SEQ ID NO: 76) FARRPTVGA  7-15 (SEQ ID NO: 77)QIPEKIQKA 16-24 (SEQ ID NO: 78) MTFGRLQGI  99-107 (SEQ ID NO: 79)ELCPPGKPT 138-146 (SEQ ID NO: 80) YVYMKRKYEA 48-57 (SEQ ID NO: 81)EAMTKLGFKA 56-65 (SEQ ID NO: 82) MTKLGFKATL 58-67 (SEQ ID NO: 83)RAEDFQGNDL 75-84 (SEQ ID NO: 84) ELCPPGKPTT 138-147 (SEQ ID NO: 85) A3TLPPFMCNK 66-74 (SEQ ID NO: 86) KIFYVYMKRK 45-54 (SEQ ID NO: 87) A24KYEAMTKLGF 54-63 (SEQ ID NO: 88) B7 RPQMTFGRL  96-104 (SEQ ID NO: 89)GPQNDGKEL 131-139 (SEQ ID NO: 90) B8 RLRERKQL 167-174 (SEQ ID NO: 91)B35 FSKEEWEKM 32-40 (SEQ ID NO: 92) B44 YEAMTKLGF 55-63 (SEQ ID NO: 93)RERKQLVIY 169-177 (SEQ ID NO: 94) B52 LQGISPKIM 104-112 (SEQ ID NO: 95)KQLVIYEEI 172-180 (SEQ ID NO: 96) SSX-1 A2 AMTKLGEKV 57-65 (SEQ ID NO:97) AMTKLGFKV 56-65 (SEQ ID NO: 98) FAKRPRDDA  7-15 (SEQ ID NO: 99)KASEKRSKA 16-24 (SEQ ID NO: 100) YVYMKRNYKA 48-57 (SEQ ID NO: 101)KAMTKLGFKV 56-65 (SEQ ID NO: 102) MTKLGFKVT 58-66 (SEQ ID NO: 103)MTKLGFKVTL 58-67 (SEQ ID NO: 104) RIQVEHPQMT  91-100 (SEQ ID NO: 105)MTFGRLHRI  99-107 (SEQ ID NO: 106) A3 TLPPFMCNK 66-74 (SEQ ID NO: 107)A24 NYKAMTKLGF 54-63 (SEQ ID NO: 108) B7 HPQMTFGRL  96-104 (SEQ ID NO:109) GPQNDGKOL 131-139 (SEQ ID NO: 110) B8 RLRERKQL 167-174 (SEQ ID NO:111) B44 RERKQLVIY 169-177 (SEQ ID NO: 112) B52 KQLVIYEEI 172-180 (SEQID NO: 113) MTFGRLHRII  99-108 (SEQ ID NO: 114) NY- A2 SISSCLQQL 148-156(SEQ ID NO: 115) GTGGSTGDA  7-15 (SEQ ID NO: 116) RASGPGGGA 52-60 (SEQID NO: 117) GARGPESRL 79-87 (SEQ ID NO: 118) ATPMEAELA  97-105 (SEQ IDNO: 119) FTVSGNILT 126-134 (SEQ ID NO: 120) LTAADHRQL 137-145 (SEQ IDNO: 121) QLSLLMWIT 155-163 (SEQ ID NO: 122) LMWITQCFL 159-167 (SEQ IDNO: 123) FATPMEAEL  96-104 (SEQ ID NO: 124) TVSGNILTI 127-135 (SEQ IDNO: 125) ATGGRGPRGA 39-48 (SEQ ID NO: 126) GAPRGPHGGA 59-68 (SEQ ID NO:127) LARRSLAQDA 104-113 (SEQ ID NO: 128) ITQCFLPVFL 162-171 (SEQ ID NO:129)

The foregoing examples describe the isolation and cloning of nucleicacid molecules for the SSX4, splice variant of SSX4, and SSX5 genes aswell as methods for determining expression of the various SSX genes as apossible indication of cancer. As was indicated, supra, these genes areexpressed in tumor cells, thereby enabling the skilled artisan toutilize these for, e.g., assaying for cancer. The determination ofexpression can be carried out via, e.g., determination of transcripts ofan SSX gene or genes, via nucleic acid hybridization, such as viapolymerase chain reaction. In a preferred embodiment, one determinespresence of a transcript of an SSX gene by contacting a sample with anucleic acid molecule which specifically hybridizes to the transcript.

The hybridization of the nucleic acid molecule to a target is indicativeof expression of an SSX gene, and of the possibility of cancer.Preferably, this is done with two primer molecules, as in a polymerasechain reaction. Determination of expression of more than one SSX gene inthe context by these assays also a part of the invention. For theconvenience of the artisan, the nucleotide sequences of SSX1 and SSX2,which are known, are presented herein as SEQ ID NOS: 1 & 2.

Alternate assays are also a part of the invention. Members of the CTfamily are known to provoke antibodies in the individual who expresses aCT family member. Hence, one can carry out the assays described hereinvia, e.g., determining antibodies in a sample taken from a subject inquestion. Most preferably, the sample being analyzed is serum. Suchassays can be carried out in any of the standard ways one determinesantibodies, such as by contacting the sample with an amount of proteinor proteins, and any additional reagents necessary to determine whetheror not the antibody binds. One approach involves the use of immobilizedprotein, where the protein is immobilized in any of the standard waysknown to the art, followed by contact with the sample and then, e.g.,anti-IgG, anti-Fc antibodies, and so forth. Conversely, presence of anSSX protein can also be determined, using antibodies in the place of theproteins of the above described assays.

The correlation of SSX expression with cancer also suggests varioustherapeutic methods and compositions useful in treating conditionsassociated with abnormal SSX expression. “Abnormal SSX expression” inthis context may mean expression per se, or levels which differ fromthose in a normal individual, i.e., they may be lower or higher.

The invention envisions therapeutic approaches such as the use ofantisense molecules to inhibit or block expression. This antisensemolecules are oligonucleotides which hybridize to the nucleic acidmolecules and inhibit their expression. Preferably these are 17-50nucleotides in length. These antisense oligonucleotides are preferablyadministered in combination with a suitable carrier, such as a cationicliposome.

Other therapeutic approaches include the administration of SSX proteinsper se, one or more antigenic peptides derived therefrom, as well asso-called polytopic vaccines. These include a plurality of antigenicpeptides, untied together, preferably by linker sequences. The resultingpeptides may bind to either MHC-Class I or Class II molecules. Theseproteins, peptides, or polytopic vaccines may be administered incombination with an appropriate adjuvant. They may also be administeredin the form of genetic constructs which are designed to permitexpression of the protein, the peptide, the polytopic structures, etc.Peptides and polytopic structures can be expressed by so-called“minigenes” i.e., DNA molecules designed to express portions of theentire SSX molecule, or the various portions of the molecules, linkedtogether as described supra. One can formulate the therapeuticcompositions and approaches described herein such that one, or more thanone SSX protein, is used as the source of the compositions. In otherwords, if a whole protein approach is used, one SSX molecule may beused, or two or more may be combined in one formulation. For peptides,these can all be taken from one SSX molecule, or be combinations ofpeptides taken from more than one. The polytopic structures describedherein can also be made up of components of one, or more than one, SSXmolecule.

The amount of agent administered and the manner in which it isadministered will, vary, based on the condition being treated and theindividual. Standard forms of administration, such as intravenous,intradermal, subcutaneous, oral, rectal and transdermal administrationcan be used. With respect to formulations, the proteins and or peptidesmay be combined with adjuvant and/or carriers such as a saponin, GM-CSF,one or more interleukin, an emulsifying oil such as vitamin E, one ormore heat shock protein, etc.

When the nucleic acid approach is utilized, various vectors, such asVaccinia or adenovirus based vectors can be used. Any vector useful ineukaryotic transfection, such as in transfection of human cells, can beused. These vectors can be used to produce, e.g., cells such asdendritic cells which present relevant peptide/MHC complexes on theirsurface. The cells can then be rendered non-proliferative prior to theiradministration, using standard methodologies.

Also a part of the invention are peptides which consist of amino acidsequences corresponding to portions of SSX molecules, or the NY-ESO-1molecule, such as those peptide sequences described supra. As has beenshown, such peptides bind to MHC molecules, such as HLA-A2 molecules,and provoke proliferation of cytolytic T cells against the formedcomplexes. As it has been shown that cells which express the full lengthmolecules (NY-ESO-1, or SSX molecules) are in fact recognized by CTLswhich were generated following pulsing of cells with relevant peptides.This result indicates that both the peptides and CTLs should be usefultherapeutic agents. Hence, an additional aspect of the invention is theadministration of one or more peptides, derived from NY-ESO-1 or an SSXmolecule as described, alone or in combination, such as in antigen“cocktails.” Such cocktails can include a mixture of peptides, whichhave been formulated following typing of a particular patient's HLAtype. Similarly, CTLs, developed in vitro, can be administered to thepatient, in view of the recognition that the peptides are presentedfollowing endogenous expression of the full length molecule.

It is to be pointed out that when an MHC molecule is mentioned, such asHLA-A2, this is meant to include all allelic forms of that molecule.There are various types of HLA-A2 molecules which are known, and whilethese differ in a few amino acids, the degree of disparity is generallyless than 10 amino acids over the full length of the molecule, and thedifferences are not expected to impact the ability of the form of themolecule to bind to peptides. Hence, a peptide which binds to anHLA-A*0201 molecule may by presumed to also bind to HLA-A*0202,HLA-A*0204, HLA-A*0205, HLA-A*0206, HLA-A*0207, HLA-A*0209, and soforth.

Other aspects of the invention will be clear to the skilled artisan andneed not be reiterated herein.

The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention in the useof such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, it being recognizedthat various modifications are possible within the scope of theinvention.

131 1 766 DNA Homo sapiens 1 cactttgtca ccaactgctg ccaactcgcc accactgctgccgcaatcgc aaccactgct 60 ttgtctctga agtgagactg ctcctggtgc catgaacggagacgacacct ttgcaaagag 120 acccagggat gatgctaaag catcagagaa gagaagcaaggcctttgatg atattgccac 180 atacttctct aagaaagagt ggaaaaagat gaaatactcggagaaaatca gctatgtgta 240 tatgaagaga aactataagg ccatgactaa actaggtttcaaagtcaccc tcccaccttt 300 catgtgtaat aaacaggcca cagacttcca ggggaatgattttgataatg accataaccg 360 caggattcag gttgaacatc ctcagatgac tttcggcaggctccacagaa tcatcccgaa 420 gatcatgccc aagaagccag cagaggacga aaatgattcgaagggagtgt cagaagcatc 480 tggcccacaa aacgatggga aacaactgca ccccccaggaaaagcaaata tttctgagaa 540 gattaataag agatctggac ccaaaagggg gaaacatgcctggacccaca gactgcgtga 600 gagaaagcag ctggtgattt atgaagagat cagtgaccctgaggaagatg acgagtaact 660 cccctggggg atacgacaca tgcccttgat gagaagcagaacgtggtgac ctttcacgaa 720 catgggcatg gctgcggctc cctcgtcatc aggtgcatagcaagtg 766 2 931 DNA Homo sapiens 2 actttctctc tctttcgatt cttccatactcagagtacgc acggtctgat tttctctttg 60 gattcttcca aaatcagagt cagactgctcccggtgccat gaacggagac gacgcctttg 120 caaggagacc cacggttggt gctcaaataccagagaagat ccaaaaggcc ttcgatgata 180 ttgccaaata cttctctaag gaagagtgggaaaagatgaa agcctcggag aaaatcttct 240 atgtgtatat gaagagaaag tatgaggctatgactaaact aggtttcaag gccaccctcc 300 cacctttcat gtgtaataaa cgggccgaagacttccaggg gaatgatttg gataatgacc 360 ctaaccgtgg gaatcaggtt gaacgtcctcagatgacttt cggcaggctc cagggaatct 420 ccccgaagat catgcccaag aagccagcagaggaaggaaa tgattcggag gaagtgccag 480 aagcatctgg cccacaaaat gatgggaaagagctgtgccc cccgggaaaa ccaactacct 540 ctgagaagat tcacgagaga tctggacccaaaagggggga acatgcctgg acccacagac 600 tgcgtgagag aaaacagctg gtgatttatgaagagatcag cgaccctgag gaagatgacg 660 agtaactccc ctcagggata cgacacatgcccatgatgag aagcagaacg tggtgacctt 720 tcacgaacat gggcatggct gcggacccctcgtcatcagg tgcatagcaa gtgaaagcaa 780 gtgttcacaa cagtgaaaag ttgagcgtcatttttcttag tgtgccaaga gttcgatgtt 840 agcgtttacg ttgtattttc ttacactgtgtcattctgtt agatactaac atttcattga 900 tgacgaagac atacttaatc gatatttggt t931 3 23 DNA Homo sapiens 3 cacacaggat ccatgaacgg aga 23 4 33 DNA Homosapiens 4 cacacaaagc tttgagggga gttactcgtc atc 33 5 576 DNA Homo sapiens5 atgaacggag acgacgcctt tgcaaggaga cccagggatg atgctcaaat atcagagaag 60ttacgaaagg ccttcgatga tattgccaaa tacttctcta agaaagagtg ggaaaagatg 120aaatcctcgg agaaaatcgt ctatgtgtat atgaagctaa actatgaggt catgactaaa 180ctaggtttca aggtcaccct cccacctttc atgcgtagta aacgggctgc agacttccac 240gggaatgatt ttggtaacga tcgaaaccac aggaatcagg ttgaacgtcc tcagatgact 300ttcggcagcc tccagagaat cttcccgaag atcatgccca agaagccagc agaggaagaa 360aatggtttga aggaagtgcc agaggcatct ggcccacaaa atgatgggaa acagctgtgc 420cccccgggaa atccaagtac cttggagaag attaacaaga catctggacc caaaaggggg 480aaacatgcct ggacccacag actgcgtgag agaaagcagc tggtggttta tgaagagatc 540agcgaccctg aggaagatga cgagtaactc ccctcg 576 6 576 DNA Homo sapiens 6atgaacggag acgacgcctt tgtacggaga cctagggttg gttctcaaat accacagaag 60atgcaaaagg ccttcgatga tattgccaaa tacttctctg agaaagagtg ggaaaagatg 120aaagcctcgg agaaaatcat ctatgtgtat atgaagagaa agtatgaggc catgactaaa 180ctaggtttca aggccaccct cccacctttc atgcgtaata aacgggtcgc agacttccag 240gggaatgatt ttgataatga ccctaaccgt gggaatcagg ttgaacatcc tcagatgact 300ttcggcaggc tccagggaat cttcccgaag atcacgcccg agaagccagc agaggaagga 360aatgattcaa agggagtgcc agaagcatct ggcccacaga acaatgggaa acagctgcgc 420ccctcaggaa aactaaatac ctctgagaag gttaacaaga catctggacc caaaaggggg 480aaacatgcct ggacccacag agtgcgtgag agaaagcaac tggtggatta tgaagagatc 540agcgaccctg cggaagatga cgagtaactc ccctca 576 7 24 DNA Homo sapiens 7ctaaagccat gcagagaagg aagc 24 8 25 DNA Homo sapiens 8 agatctcttattaatcttcc agaaa 25 9 23 DNA Homo sapiens 9 gtgctcaaat accagagaag atc 2310 23 DNA Homo sapiens 10 ttttgggtcc agatctcctc gtg 23 11 24 DNA Homosapiens 11 ggaagagtgg gaaaagatga aagt 24 12 22 DNA Homo sapiens 12ccccttttgg gtccagatat ca 22 13 25 DNA Homo sapiens 13 aaatcgtctatgtgtatatg aagct 25 14 22 DNA Homo sapiens 14 gggtcgctga tctcttcata ac22 15 23 DNA Homo sapiens 15 gttctcaaat accacagaag atg 23 16 20 DNA Homosapiens 16 ctctgctggc ttctcgggcg 20 17 27 DNA Homo sapiens 17 acagcattaccaaggacagc agccacc 27 18 27 DNA Homo sapiens 18 gccaacagca agatgcataccagggac 27 19 9 PRT Homo sapiens 19 Arg Leu Leu Glu Phe Tyr Leu Ala Met1 5 20 9 PRT Homo sapiens 20 Ser Leu Ala Gln Asp Ala Pro Pro Leu 1 5 219 PRT Homo sapiens 21 Ser Thr Leu Glu Lys Ile Asn Lys Thr 1 5 22 9 PRTHomo sapiens 22 Lys Ala Ser Glu Lys Ile Ile Tyr Val 1 5 23 9 PRT Homosapiens 23 Asp Ala Phe Val Arg Arg Pro Arg Val 1 5 24 10 PRT Homosapiens 24 Gln Ile Pro Gly Gln Lys Met Gln Lys Ala 1 5 10 25 9 PRT Homosapiens 25 Met Thr Lys Leu Gly Phe Lys Ala Thr 1 5 26 9 PRT Homo sapiens26 Met Thr Phe Gly Arg Leu Gln Gly Ile 1 5 27 9 PRT Homo sapiens 27 AsnThr Ser Glu Lys Val Asn Lys Thr 1 5 28 10 PRT Homo sapiens 28 Tyr ValThr Met Lys Arg Lys Tyr Glu Ala 1 5 10 29 10 PRT Homo sapiens 29 Tyr MetLys Arg Lys Tyr Glu Ala Met Thr 1 5 10 30 10 PRT Homo sapiens 30 Glu AlaMet Thr Lys Leu Gly Phe Lys Ala 1 5 10 31 10 PRT Homo sapiens 31 Met ThrLys Leu Gly Phe Lys Ala Thr Leu 1 5 10 32 10 PRT Homo sapiens 32 Arg LeuGln Gly Ile Gly Pro Lys Ile Thr 1 5 10 33 10 PRT Homo sapiens 33 Gln LeuAla Pro Ser Gly Lys Leu Asn Thr 1 5 10 34 10 PRT Homo sapiens 34 Gly IlePhe Pro Lys Ile Thr Pro Glu Leu 1 5 10 35 10 PRT Homo sapiens 35 Lys LeuAsn Thr Ser Glu Lys Val Asn Lys 1 5 10 36 10 PRT Homo sapiens 36 Lys TyrGlu Ala Met Thr Lys Leu Gly Phe 1 5 10 37 9 PRT Homo sapiens 37 His ProGln Met Thr Phe Gly Arg Leu 1 5 38 9 PRT Homo sapiens 38 Gly Pro Gln AsnAsn Gly Lys Gln Leu 1 5 39 8 PRT Homo sapiens 39 Arg Val Arg Glu Arg LysGln Leu 1 5 40 9 PRT Homo sapiens 40 Tyr Glu Ala Met Thr Lys Leu Gly Phe1 5 41 9 PRT Homo sapiens 41 Arg Glu Arg Lys Gln Leu Val Ile Tyr 1 5 429 PRT Homo sapiens 42 Lys Gln Leu Val Ile Tyr Glu Glu Ile 1 5 43 10 PRTHomo sapiens 43 Met Thr Phe Gly Arg Leu Gln Gly Ile Phe 1 5 10 44 9 PRTHomo sapiens 44 Lys Ser Ser Glu Lys Ile Val Tyr Val 1 5 45 9 PRT Homosapiens 45 Val Met Thr Lys Leu Gly Phe Lys Val 1 5 46 10 PRT Homosapiens 46 Tyr Val Tyr Met Lys Leu Asn Tyr Glu Val 1 5 10 47 10 PRT Homosapiens 47 Lys Leu Asn Tyr Glu Val Met Thr Lys Leu 1 5 10 48 9 PRT Homosapiens 48 Phe Ala Arg Arg Pro Arg Asp Asp Ala 1 5 49 9 PRT Homo sapiens49 Gln Ile Ser Glu Lys Leu Arg Lys Ala 1 5 50 9 PRT Homo sapiens 50 MetThr Phe Gly Ser Leu Gln Arg Ile 1 5 51 9 PRT Homo sapiens 51 Ser Leu GlnArg Ile Phe Pro Lys Ile 1 5 52 9 PRT Homo sapiens 52 Lys Ile Val Tyr ValTyr Met Lys Leu 1 5 53 9 PRT Homo sapiens 53 Lys Leu Arg Lys Ala Phe AspAsp Ile 1 5 54 10 PRT Homo sapiens 54 Lys Leu Arg Lys Ala Phe Asp AspIle Ala 1 5 10 55 10 PRT Homo sapiens 55 Tyr Met Lys Leu Asn Tyr Glu ValMet Thr 1 5 10 56 10 PRT Homo sapiens 56 Met Thr Lys Leu Gly Phe Lys ValThr Leu 1 5 10 57 10 PRT Homo sapiens 57 Gln Leu Cys Pro Pro Gly Asn ProSer Thr 1 5 10 58 9 PRT Homo sapiens 58 Lys Leu Asn Tyr Glu Val Met ThrLys 1 5 59 10 PRT Homo sapiens 59 Asn Tyr Glu Val Met Thr Lys Leu GlyPhe 1 5 10 60 9 PRT Homo sapiens 60 Arg Pro Gln Met Thr Phe Gly Ser Leu1 5 61 9 PRT Homo sapiens 61 Lys Pro Ala Glu Glu Glu Asn Gly Leu 1 5 629 PRT Homo sapiens 62 Gly Pro Gln Asn Asp Gly Lys Gln Leu 1 5 63 9 PRTHomo sapiens 63 Cys Pro Pro Gly Asn Pro Ser Thr Leu 1 5 64 8 PRT Homosapiens 64 Arg Leu Arg Glu Arg Lys Gln Leu 1 5 65 8 PRT Homo sapiens 65Arg Pro Arg Asp Asp Ala Gln Ile 1 5 66 9 PRT Homo sapiens 66 Lys Pro AlaGlu Glu Glu Asn Gly Leu 1 5 67 9 PRT Homo sapiens 67 Tyr Glu Val Met ThrLys Leu Gly Phe 1 5 68 9 PRT Homo sapiens 68 Arg Glu Arg Lys Gln Leu ValVal Tyr 1 5 69 9 PRT Homo sapiens 69 Lys Gln Leu Val Val Tyr Glu Glu Ile1 5 70 10 PRT Homo sapiens 70 Met Thr Phe Gly Ser Leu Gln Arg Ile Phe 15 10 71 9 PRT Homo sapiens 71 Lys Ile Gln Lys Ala Phe Asp Asp Ile 1 5 729 PRT Homo sapiens 72 Lys Ala Ser Glu Lys Ile Phe Tyr Val 1 5 73 9 PRTHomo sapiens 73 Ala Met Thr Lys Leu Gly Phe Lys Ala 1 5 74 9 PRT Homosapiens 74 Arg Leu Gln Gly Ile Ser Pro Lys Ile 1 5 75 9 PRT Homo sapiens75 Arg Leu Arg Glu Arg Lys Gln Leu Val 1 5 76 9 PRT Homo sapiens 76 AspAla Phe Ala Arg Arg Pro Thr Val 1 5 77 9 PRT Homo sapiens 77 Phe Ala ArgArg Pro Thr Val Gly Ala 1 5 78 9 PRT Homo sapiens 78 Gln Ile Pro Glu LysIle Gln Lys Ala 1 5 79 9 PRT Homo sapiens 79 Met Thr Phe Gly Arg Leu GlnGly Ile 1 5 80 9 PRT Homo sapiens 80 Glu Leu Cys Pro Pro Gly Lys Pro Thr1 5 81 10 PRT Homo sapiens 81 Tyr Val Tyr Met Lys Arg Lys Tyr Glu Ala 15 10 82 10 PRT Homo sapiens 82 Glu Ala Met Thr Lys Leu Gly Phe Lys Ala 15 10 83 10 PRT Homo sapiens 83 Met Thr Lys Leu Gly Phe Lys Ala Thr Leu 15 10 84 10 PRT Homo sapiens 84 Arg Ala Glu Asp Phe Gln Gly Asn Asp Leu 15 10 85 10 PRT Homo sapiens 85 Glu Leu Cys Pro Pro Gly Lys Pro Thr Thr 15 10 86 9 PRT Homo sapiens 86 Thr Leu Pro Pro Phe Met Cys Asn Lys 1 5 8710 PRT Homo sapiens 87 Lys Ile Phe Tyr Val Tyr Met Lys Arg Lys 1 5 10 8810 PRT Homo sapiens 88 Lys Tyr Glu Ala Met Thr Lys Leu Gly Phe 1 5 10 899 PRT Homo sapiens 89 Arg Pro Gln Met Thr Phe Gly Arg Leu 1 5 90 9 PRTHomo sapiens 90 Gly Pro Gln Asn Asp Gly Lys Glu Leu 1 5 91 8 PRT Homosapiens 91 Arg Leu Arg Glu Arg Lys Gln Leu 1 5 92 9 PRT Homo sapiens 92Phe Ser Lys Glu Glu Trp Glu Lys Met 1 5 93 9 PRT Homo sapiens 93 Tyr GluAla Met Thr Lys Leu Gly Phe 1 5 94 9 PRT Homo sapiens 94 Arg Glu Arg LysGln Leu Val Ile Tyr 1 5 95 9 PRT Homo sapiens 95 Leu Gln Gly Ile Ser ProLys Ile Met 1 5 96 9 PRT Homo sapiens 96 Lys Gln Leu Val Ile Tyr Glu GluIle 1 5 97 9 PRT Homo sapiens 97 Ala Met Thr Lys Leu Gly Glu Lys Val 1 598 9 PRT Homo sapiens 98 Ala Met Thr Lys Leu Gly Phe Lys Val 1 5 99 9PRT Homo sapiens 99 Phe Ala Lys Arg Pro Arg Asp Asp Ala 1 5 100 9 PRTHomo sapiens 100 Leu Ala Ser Glu Lys Arg Ser Lys Ala 1 5 101 10 PRT Homosapiens 101 Tyr Val Tyr Met Lys Arg Asn Tyr Lys Ala 1 5 10 102 10 PRTHomo sapiens 102 Lys Ala Met Thr Lys Leu Gly Phe Lys Val 1 5 10 103 9PRT Homo sapiens 103 Met Thr Lys Leu Gly Phe Lys Val Thr 1 5 104 10 PRTHomo sapiens 104 Met Thr Lys Leu Gly Phe Lys Val Thr Leu 1 5 10 105 10PRT Homo sapiens 105 Arg Ile Gln Val Glu His Pro Gln Met Thr 1 5 10 1069 PRT Homo sapiens 106 Met Thr Phe Gly Arg Leu His Arg Ile 1 5 107 9 PRTHomo sapiens 107 Thr Leu Pro Pro Phe Met Cys Asn Lys 1 5 108 10 PRT Homosapiens 108 Asn Tyr Lys Ala Met Thr Lys Leu Gly Phe 1 5 10 109 9 PRTHomo sapiens 109 His Pro Gln Met Thr Phe Gly Arg Leu 1 5 110 9 PRT Homosapiens 110 Gly Pro Gln Asn Asp Gly Lys Gln Leu 1 5 111 8 PRT Homosapiens 111 Arg Leu Arg Glu Arg Lys Gln Leu 1 5 112 9 PRT Homo sapiens112 Arg Glu Arg Lys Gln Leu Val Ile Tyr 1 5 113 9 PRT Homo sapiens 113Lys Gln Leu Val Ile Tyr Glu Glu Ile 1 5 114 10 PRT Homo sapiens 114 MetThr Phe Gly Arg Leu His Arg Ile Ile 1 5 10 115 9 PRT Homo sapiens 115Ser Ile Ser Ser Cys Leu Gln Gln Leu 1 5 116 9 PRT Homo sapiens 116 GlyThr Gly Gly Ser Thr Gly Asp Ala 1 5 117 9 PRT Homo sapiens 117 Arg AlaSer Gly Pro Gly Gly Gly Ala 1 5 118 9 PRT Homo sapiens 118 Gly Ala ArgGly Pro Glu Ser Arg Leu 1 5 119 9 PRT Homo sapiens 119 Ala Thr Pro MetGlu Ala Glu Leu Ala 1 5 120 9 PRT Homo sapiens 120 Phe Thr Val Ser GlyAsn Ile Leu Thr 1 5 121 9 PRT Homo sapiens 121 Leu Thr Ala Ala Asp HisArg Gly Leu 1 5 122 9 PRT Homo sapiens 122 Gln Leu Ser Leu Leu Met TrpIle Thr 1 5 123 9 PRT Homo sapiens 123 Leu Met Trp Ile Thr Gln Cys PheLeu 1 5 124 9 PRT Homo sapiens 124 Phe Ala Thr Pro Met Glu Ala Glu Leu 15 125 9 PRT Homo sapiens 125 Thr Val Ser Gly Asn Ile Leu Thr Ile 1 5 12610 PRT Homo sapiens 126 Ala Thr Gly Gly Arg Gly Pro Arg Gly Ala 1 5 10127 10 PRT Homo sapiens 127 Gly Ala Pro Arg Gly Pro His Gly Gly Ala 1 510 128 10 PRT Homo sapiens 128 Leu Ala Arg Arg Ser Leu Ala Gln Asp Ala 15 10 129 10 PRT Homo sapiens 129 Ile Thr Gln Cys Phe Leu Pro Val Phe Leu1 5 10 130 11 PRT Homo sapiens 130 Ser Leu Leu Met Trp Ile Thr Gln CysPhe Leu 1 5 10 131 9 PRT Homo sapiens 131 Ser Leu Leu Met Trp Ile ThrGln Cys 1 5

What is claimed is:
 1. A method for stimulating proliferation ofcytolytic T lymphocytes comprising combining a cell which presents anHLA-A2 molecule on its surface with an isolated peptide, which consistsof the sequence of ArgLeuLeuGluPheTyrLeuAlaMet (SEQ ID NO: 19) and a Tlymphocyte containing sample of cells from an HLA-A2 positive subjecthaving melanoma, under conditions favoring formation of a complexbetween said HLA-A2 molecule and said peptide, and proliferation ofcytolytic T lymphocytes specific for said complex.
 2. The method ofclaim 1, comprising combining said cell, said peptide, and said Tlymphocyte containing sample in vitro.
 3. A method for stimulatingproliferation of cytolytic T lymphocytes in an HLA-A2 positive subjecthaving melanoma comprising administering a peptide, which consists ofthe sequence of ArgLeuLeuGluPheTyrLeuAlaMet (SEQ ID NO: 19), to saidsubject to stimulate proliferation of cytolytic T lymphocytes specificfor a complex of HLA-A2 and said peptide.
 4. The method of claim 3,wherein said peptide is administered with an adjuvant.
 5. The method ofclaim 3, wherein said peptide is administered with an interleukin.